Pig and Cow Blood During Cold Storage in CPDA-1 Solution: Hematology and Fluid Behavior

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article offers valuable insights into the storage properties of bovine and porcine blood, emphasizing the superior shelf life of bovine blood, which remains stable throughout the storage period. In contrast, porcine blood demonstrates early hemolysis. The study provides critical information for advancing blood storage practices and is supported by meticulously conducted statistical analysis.

However, three critical issues require attention:

1. The authors have not provided an in-depth discussion of the underlying physical mechanisms. The discussion should be expanded to go beyond a simple presentation of results, offering a more comprehensive exploration of the factors driving the observed phenomena.
2. The study should experimentally investigate the thixotropic responses of bovine and porcine blood. Furthermore, if feasible, employing thixo-elastoviscoplastic models (e.g., as discussed by Giannokostas et al. in Materials and Beris et al. in Soft Matter) could offer a robust framework for evaluating and interpreting the rheological behavior of the samples.
3. Lastly, the inclusion of a table summarizing the pros and cons of each type of blood would enhance the clarity and practical utility of the findings. Such a table could effectively highlight key differences in properties, shelf life, and potential applications.

Comments on the Quality of English Language

The quality of English is good but could be further refined to enhance flow and conciseness. Please ensure consistent use of the terms bovine and porcine instead of mixing them with "cow" and "pig," unless there is a specific and significant difference between the blood of female bovines and that of bulls or calves. Additionally, could you clarify what is meant by the "elastic halo"? Are you referring to the endothelial surface layer, or is it a distinct structure?

Author Response

The article offers valuable insights into the storage properties of bovine and porcine blood, emphasizing the superior shelf life of bovine blood, which remains stable throughout the storage period. In contrast, porcine blood demonstrates early hemolysis. The study provides critical information for advancing blood storage practices and is supported by meticulously conducted statistical analysis.

Reply: we thank the reviewer very much for the time, she/he spent on our manuscript and the helpful and very welcome comments. We have addressed them all in the revised version

However, three critical issues require attention:

1. The authors have not provided an in-depth discussion of the underlying physical mechanisms. The discussion should be expanded to go beyond a simple presentation of results, offering a more comprehensive exploration of the factors driving the observed phenomena.

Reply: We fully agree that the clarification of the underlying physical mechanism is of significant interest to the reader. We have added new text in the discussion section highlighting the importance of membrane reinforcement at the “cost” of lower deformability (page 13), and the issue of sample condensation when bovine blood was adjusted to the HCT of pigs (an additional study group was added), also the importance of a suspension behavior towards a Newtonian plateau. We also hope that the Conclusion section provides ideas concerning the shelf life of animal blood.

1. The study should experimentally investigate the thixotropic responses of bovine and porcine blood. Furthermore, if feasible, employing thixo-elastoviscoplastic models (e.g., as discussed by Giannokostas et al. in Materials and Beris et al. in Soft Matter) could offer a robust framework for evaluating and interpreting the rheological behavior of the samples.

Reply: It is indeed a very good idea to compare the blood of different species and we thank the reviewer for this comment. However, I am not sure if the model applies also if masses of hemoglobin are present in the suspending medium, and the RBCs do no longer have their physiological shape and functionality. We rather like to do so with our data obtained from fresh blood in a further work. Thank you for this suggestion!

1. Lastly, the inclusion of a table summarizing the pros and cons of each type of blood would enhance the clarity and practical utility of the findings. Such a table could effectively highlight key differences in properties, shelf life, and potential applications.

Reply: we added Table 2 at the end of the text

Reviewer 2 Report

Comments and Suggestions for Authors

With great interest I have read the manuscript; Pig and cow blood during cold storage in CPDA-1 solution: hematology and fluid behavior from the authors Ursula Windberger  and Andreas Sparer. I find the subject interesting and it is great to have information about RBC`s of different species. However, it is observational with small groups and the variability within the groups makes it hard to draw conclusions. For now, the data is in line with lifespan of RBC`s between pig and cow? Can anything said when loss of functionality starts? The idea is solid however, I would suggest the authors to give this subject a little more attention in increasing groups and implement controls.

 

Minor issues

-          CPDA-1 is never full out written. Citrate-Phosphate-Dextrose-Adenine-1 (CDPA-1). This also accounts for other abbreviations throughout the paper.

-          Legends state Median+/-interquartile distance. distance should be range (IQR)).

-          Statistics: that is a bit of a puzzle. Mean+/-SD is used and  Median+/-interquartile ranges (not mentioned in the statistics). The latter is not stated in the statistics part. Did you test for normal distribution? (not mentioned). It’s a mixture of parametric and non-parametric testing on the same groups.

-          Table 1, Samples sizes are really small. The spread in the cow RBC group is relatively high. If numbers were increased to 20-25 you can question if the diversity with porcine cells and the non-significance sticks. The white blood cells and platelets are really close, even in these small groups.

-          Table 1: Explain T/L and G/L in legend

-          Were samples measured in duplicates?

-          Figure 1. What was the magnification?

-          Figure 1. Error bar should be scale bar

-          Figure 5a. seems incomplete. It started on different days. Day 3 in cow and day 0 in pigs. And the frequency is missing points in the cow group.

-          Figure 6. Pig smear doesn`t have a scalebar or magnification.

-          Where samples tested for infections? Can sterility be guaranteed in syringes for 30 days?

-          Pig smears were from a different group included later in time? Did you only checked 3 pigs and 3 cows under the microscope?

 

 

 

 

 

 

Major issues:

 

-          Groups are actually too small to say anything about functional behavior.

-          The MCHC increases, however the hemolysis also significantly increased in pigs. Isn`t that contradictory?

-           Bright field images are hard to determine. Do you have higher magnification? Explain what you think is a ghost cell or crenated cells. Average size of a human RBC is about 5um. These cells appear a lot smaller. When doing microscopy, make sure to get some experienced help. These seem, wet mounts at 200x? There are simple stains you can use  for better determination of reb blood cells.

-          Figure 2. most of your debris is most likely from WBC and Platelets. The reduction in red blood cells is minimal and in line with the overall quantity and lifespan of pig and cow?

-          The authors address hemolysis measurements and that capillaries were photographed, however these were not included. Why?

-          Lot of graphs show high variability. Why not increase groups numbers?

-          The hematocrit of pigs is a lot higher. Why did the authors not equalize hematocrit? I would have used a diluted sample of pigs blood to cow hematocrit as a 3^rd group.

-          Why not include 50ml syringes of human blood? And why syringes?

-          The biggest issue is that the authors have something that is completely observational however, the biggest interest is were the RBC loses its functionality? Deformability/aggregation/oxygen binding ext.

-          In the discussion, 294, the authors address the low permeability of porcine RBC`s, however, in my opinion the do seem to leak hemoglobin? As the number of red blood cells do not drop massively, but severe hemolysis was observed (data not shown)

 

Comments on the Quality of English Language

The paper needs attention. implement abbreviations. go through the paper once more from textual issues. some figures need attention.  

Author Response

With great interest I have read the manuscript; Pig and cow blood during cold storage in CPDA-1 solution: hematology and fluid behavior from the authors Ursula Windberger and Andreas Sparer. I find the subject interesting and it is great to have information about RBC`s of different species. However, it is observational with small groups and the variability within the groups makes it hard to draw conclusions. For now, the data is in line with lifespan of RBC`s between pig and cow? Can anything said when loss of functionality starts? The idea is solid however, I would suggest the authors to give this subject a little more attention in increasing groups and implement controls. 

Reply: we thank the reviewer for the time, she/he spent on our manuscript and the many helpful and very welcome comments. We hope to have addressed them clearly and distinctly in the revised version, especially we have added a third group.

 

Minor issues

-          CPDA-1 is never full out written. Citrate-Phosphate-Dextrose-Adenine-1 (CDPA-1). This also accounts for other abbreviations throughout the paper. 

Reply: has been added, thank you.

-          Legends state Median+/-interquartile distance. distance should be range (IQR)). 

Reply: has been adapted, thank you.

-          Statistics: that is a bit of a puzzle. Mean+/-SD is used and  Median+/-interquartile ranges (not mentioned in the statistics). The latter is not stated in the statistics part. Did you test for normal distribution? (not mentioned). It’s a mixture of parametric and non-parametric testing on the same groups.

Reply: in the box plots there are asterisks to indicate the mean values, displayed in the other figures. Please find below an example for a bar chart with mean and standard deviation (Fig. 5f). We think that the box plots with asterisks are better informative and we would like to keep them in the manuscript. We did not test for normal distribution. The small sample size does not allow the clarification of a normal distribution. This is why we used the Wilcoxon matched pairs signed rank test

-          Table 1, Samples sizes are really small. The spread in the cow RBC group is relatively high. If numbers were increased to 20-25 you can question if the diversity with porcine cells and the non-significance sticks. The white blood cells and platelets are really close, even in these small groups.

Reply: thank you for your comment. We shall check this in the next approach, also to clarify the kind of solidification of HCT adjusted bovine samples

-          Table 1: Explain T/L and G/L in legend

Reply: Tera per Liter and Giga per Liter are common dimensions. They do not need an explanation

-          Were samples measured in duplicates?

Reply: No

-          Figure 1. What was the magnification?

Reply: the magnification was 60-fold

-          Figure 1. Error bar should be scale bar

Reply: yes, was adapted, thank you!

-          Figure 5a. seems incomplete. It started on different days. Day 3 in cow and day 0 in pigs. And the frequency is missing points in the cow group. 

Reply: we could not do rheology on day 0 with cow samples due to technical reasons. The rheometer could not keep the temperature. We indicate this issue inow n the figure legend. Frequency sweep tests: although not looking nice, there is nothing missing. The obtained torque was below the detection limit, and displayed values are not valid and therefore deleted. We clarify this issue in the figure legend of the revised manuscript

-          Figure 6. Pig smear doesn`t have a scalebar or magnification. 

Reply: thank you! – scale bar has been added

-          Where samples tested for infections? Can sterility be guaranteed in syringes for 30 days?

Reply: yes, we tested in pigs; and no, it cannot. All pig samples were tested for infections on Columbia and Schaedler agar, identified by MALDI-TOF MS. One pig blood sample showed contamination with Corynebacterium stationis at start of the storage period, all other samples were germ-free in both aerobic and anaerobic screening after incubation at 37°C for 48 hours. After storage, all samples featured massive bacterial growth after 24 hours incubation time. Three different bacterial species were identified: Staphylococcus xylosus in eight samples, Pantoea agglomerans in four samples, Staphylococcus sciuri in one sample. Cow samples were not tested for bacteria/fungi, as we expected a likewise contamination.

-          Pig smears were from a different group included later in time? Did you only checked 3 pigs and 3 cows under the microscope? 

Reply: we checked all cows under the microscope (native and HCT adjusted samples), but only 3 pigs. The reason for this discrepancy is that we explored first the pig group and afterwards the cow group. We learnt from the cow group that RBCs maintained their shape, which was unexpected to us. In order to compare with pig, we draw blood from 3 additional pigs to compare with cow.

 

 Major issues:

-          Groups are actually too small to say anything about functional behavior.

Reply: we agree that sample size is too low to test for normal distribution, which requires min. 30 samples. We also agree that the study is purely observational, and we clarify this in the introduction section of the revised version. However, we believe that our data can draw a clear picture of what is happening during storage.  

-          The MCHC increases, however the hemolysis also significantly increased in pigs. Isn`t that contradictory?

Reply: MCHC reflects the hemoglobin concentration present in the remaining RBCs. There is no contradiction.

-           Bright field images are hard to determine. Do you have higher magnification? Explain what you think is a ghost cell or crenated cells. Average size of a human RBC is about 5um. These cells appear a lot smaller. When doing microscopy, make sure to get some experienced help. These seem, wet mounts at 200x? There are simple stains you can use for better determination of reb blood cells.

Reply: we agree and apologize: we checked only native (unstained) smears. We shall look more on adequate smears in the future and apologize. Ghost cells and crenated cells are indicated in the revised version. RBCs from most mammalian species are small than human RBCs.

-          Figure 2. most of your debris is most likely from WBC and Platelets. The reduction in red blood cells is minimal and in line with the overall quantity and lifespan of pig and cow? 

Reply: it is difficult to assume the reduction in RBC count from a blood smear. The reduction of RBC count in pig samples and the much lesser reduction of RBC count in cow samples due to storage are in line with the circulatory lifespan of pig and cow RBCs, as written in the discussion section, first paragraph. 

-          The authors address hemolysis measurements and that capillaries were photographed, however these were not included. Why?

Reply: because they do not provide more information than “yes” or “no”. Such information can be given in the text within one sentence. Enclosed are examples from stored cow blood with adjusted HCT at storage day 30. The numbers are the ear markings of the cows.

-          Lot of graphs show high variability. Why not increase groups numbers? 

Reply: we are aware of the low group number in cows, and added a further group (5 cows) with adjusted HCT (to the porcine value) to validate the findings obtained with native HCT. We found similar development with these new cows, but also one interesting feature that distinguishes the ageing process of native from HCT adjusted samples. These features are shown and discussed in the revised version.

-          The hematocrit of pigs is a lot higher. Why did the authors not equalize hematocrit? I would have used a diluted sample of pigs blood to cow hematocrit as a 3^rd group.

Reply: we add data from 5 cow samples with HCT standardization to the pig value as a third group in the revised manuscript. We adapted Figures 2 and 3, and also Fig. 5c, and describe the findings in the results and discussion section.

-          Why not include 50ml syringes of human blood? And why syringes?

Reply: because we drew blood into these syringes (see photo below). We had to find a solution to mix the correct volume of blood that we drew from the conscious animals in their familiar environment with the laid CPDA-1 in the container without opening the container. We did not obtain ethical clearance for blood drawal of 500mL to use human blood bags. Again in the lab, we did not want to change the container, because we could easily mount them on the overhead agitator. In the Conclusion section, we mention that storage containers other than 50 mL syringes may allow a better resuspension of cell clusters, but this is not proven.

-          The biggest issue is that the authors have something that is completely observational however, the biggest interest is were the RBC loses its functionality? Deformability/aggregation/oxygen binding ext. 

Reply: This is generally a rheological study to show the quality of the suspensions, combined with features from routine hematology to explain the rheological findings. This concept must be very irritating for a hematologist, and we deeply apologize. We focused on the bulk effect to measure the flow resistance and to see when the suspension “breaks apart”, which we indicate as shelf life. The hematological parameters are used to explain why the suspension changes its state. This is given by the switch towards Newtonian behavior, and is also seen in the behavior at quasi-static state. However, some RBC functionality can be extrapolated from the rheological tests, e.g. the presence of stiff RBCs explains the increase of shear viscosity at 1000s-1. To our best knowledge, this is the first rheological investigation of aging cow and pig blood, and can be the start of further investigations to look specifically on RBC function.

 -          In the discussion, 294, the authors address the low permeability of porcine RBC`s, however, in my opinion the do seem to leak hemoglobin? As the number of red blood cells do not drop massively, but severe hemolysis was observed (data not shown)

Reply: we refer to the low membrane permeability to glucose via transporter. Hemolysis occurs due to overall membrane damage. The severe hemolysis in porcine samples is indicated in the results section: “mean plasma free hemoglobin concentration was of over 200 g dL^-1 on day 30.”

Comments on the Quality of English Language

The paper needs attention. implement abbreviations. go through the paper once more from textual issues. some figures need attention.  

Reply: thank you, we agree and asked a native speaker to go through the text.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The reviewed paper presents the results of the hematology and hemorhrology properties of pig and bovine blood during storage. The paper is written quite correctly, and has great practical significance from the point of view of using such blood tests in biomedical research. When analyzing the introduction, I feel somewhat unsatisfied. I do not know quite what is this scientific novelty that should be emphasised. In addition, the purpose of the paper was not clearly formulated. I propose to extend the introduction to other similar studies.

 Additionally, I found a few more inaccuracies that need to be corrected.

 1. Figure captions should start with a capital letter

2. Why were the studies conducted at a temperature of 22 degrees.

3. Figure 2, the caption of the figure should state what figures A and B refer to.

4. The same applies to Figure 3, there are also no figures marked as a, b, c, and d

5. Some cited articles are from the previous century, such as 11, 18, 19, 22, 23, 24. Can they be replaced with more recent ones?

6. Line 305, citations (5)(27)(28)(29) should be in one bracket after a comma, the same applies to other items, e.g. lines 269, 295, 296. Please read the text carefully

 With these corrections, the work will become suitable for publication in Biophysica

 

Author Response

The reviewed paper presents the results of the hematology and hemorhrology properties of pig and bovine blood during storage. The paper is written quite correctly, and has great practical significance from the point of view of using such blood tests in biomedical research. When analyzing the introduction, I feel somewhat unsatisfied. I do not know quite what is this scientific novelty that should be emphasised. In addition, the purpose of the paper was not clearly formulated. I propose to extend the introduction to other similar studies.

Reply: we thank the reviewer for the time spent on our manuscript and the helpful comments. We have addressed them in the revised version, and we adapted the Introduction section to make our approach clearer. Thank you for this comment.

 Additionally, I found a few more inaccuracies that need to be corrected.

1. Figure captions should start with a capital letter

Reply: thank you, has been adapted

2. Why were the studies conducted at a temperature of 22 degrees.

Reply: we first performed rheometry at 22 and 37°C in pigs, but with increasing storage time, the blood became vulnerable and the tests at quasi-static conditions showed higher errors at 37°C. In the subsequent tests with cow blood, we thus decided to perform rheometry only at 22°C. This allowed us also to draw less blood from the animals (ethical concern). We need a new portion for each test and for each temperature due to plasma skimming and RBC sedimentation

3. Figure 2, the caption of the figure should state what figures A and B refer to.

Reply: thank you, has been adapted

4. The same applies to Figure 3, there are also no figures marked as a, b, c, and d

Reply: thank you, has been adapted

5. Some cited articles are from the previous century, such as 11, 18, 19, 22, 23, 24. Can they be replaced with more recent ones?

Reply: it seams that they cannot be replaced, I wish I could. I have not found recent information on the membrane composition/functionality of bovine and porcine membranes except Kennedy et al. (DOI 10.31083/j.fbl2812331). Some of these references were deleted, because there do not support the understanding of the findings

6. Line 305, citations (5)(27)(28)(29) should be in one bracket after a comma, the same applies to other items, e.g. lines 269, 295, 296. Please read the text carefully

Reply: has been corrected

 With these corrections, the work will become suitable for publication in Biophysica

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I think this is much clearer than the previous version and I think the you reached the limited what you can do. I have no further comments